Structural and Biochemical Characterization of Human NOD2 Activation and Assembly

Abstract

NOD2 is a cytosolic pattern-recognition receptor that detects bacterial muramyl dipeptide (MDP) and activates inflammatory signaling through recruitment of receptor-interacting serine/threonine-protein kinase 2 (RIPK2). Genetic variants in NOD2 are strongly associated with Crohn’s disease and Blau syndrome, yet the molecular basis of receptor activation and adaptor engagement remains incompletely defined. In particular, the architecture of activated NOD2 and its coupling to RIPK2 assembly have not been structurally resolved. Here, I establish a recombinant platform for biochemical and structural interrogation of human NOD2 and RIPK2. Full-length and domain-truncated NOD2 constructs were expressed in insect cells and purified for downstream analysis. Biochemical and imaging experiments showed that NOD2 undergoes temperature- and nucleotide-dependent oligomerization in vitro. Negative-stain electron microscopy revealed formation of discrete rounded oligomeric particles following ATPγS incubation, whereas size-exclusion chromatography and mass photometry indicated that both full-length and ΔCARD NOD2 were predominantly monomeric under resting conditions, with minor higher-order populations. The ΔCARD construct exhibited improved biochemical homogeneity and yielded a high-resolution cryo-electron microscopy reconstruction of the NOD2 monomeric core. This structure showed strong agreement with computational predictions for the NACHT and LRR regions and provides a framework for future analysis of disease-associated mutations and ligand-responsive conformational change. In contrast, oligomeric assemblies of both full-length and ΔCARD NOD2 remained structurally heterogeneous, limiting high-resolution reconstruction of the activated state. RIPK2 was also reconstituted in vitro and shown to form filamentous assemblies after tag removal. Biochemical assays, including crosslinking and native gel analysis, supported physical association between NOD2 and RIPK2, although a stable and structurally defined signaling supercomplex was not resolved. In THP-1 macrophages, MDP stimulation promoted redistribution of NOD2 toward a membrane-associated fraction that remained recoverable after detergent extraction, consistent with activation-dependent spatial reorganization. Together, these findings establish a tractable biochemical and structural framework for studying NOD2 activation. The data support a model in which NOD2 contains an intrinsic assembly program within its NACHT-LRR core, while RIPK2 engagement and higher-order signaling complex formation remain condition-dependent and structurally unresolved. This work provides a foundation for future studies of ligand binding, adaptor recruitment, disease-associated mutation mechanisms, and therapeutic modulation of the NOD2 pathway.